Embodiments of the inventive concept described herein relate to a small molecule detection and localization method based on Matrix-Assisted Laser Desorption Ionization (MALDI) imaging mass spectrometry, and more particularly, relate to a method for detecting whether small molecules are bound with a target protein and for measuring a binding distribution between the small molecules and the target protein in a cell or in a biosample including organelles by comparing a result of MALDI mass spectrometry with a result of immunofluorescence of the small molecules, which are used as drugs, for the target protein, and for determining as angiogenesis is inhibited in a portion overlapping with a portion where the drug small molecules are present after the MALDI mass spectrometry in the cell or in the biosample including organelles and a portion where the target protein is present after immunofluorescence, as well as for detecting presence or absence and a distribution state of small molecules used as drugs in a sample by using MALDI mass spectrometry.
In general, a mass spectrometer is a spectrometer for measuring mass of a compound, determining a molecular weight of a compound by measuring mass-to-charge quantity after electrically charging and ionizing a compound. Since the first use of mass spectrometry in the early 1900s, various ionization methods have been developed and proposed for mass spectrometry of nonvolatile or thermo-unstable materials, for example, including SIMS, FD, FAB, MALDI, and so on.
The histology-directed Matrix-Assisted Laser Desorption Ionization (MALDI) imaging mass spectrometry developed by Richard M. Caprioli is an imaging mass spectrometry using MALDI, obtaining mass spectrometry information directly from a tissue through MALDI by coating a matrix on the surface of the tissue which is to be inspected.
While the beginning of applying MALDI directly to a human anatomy is recent years, there has been a report that inspecting a frozen tissue by using sinapinic acid as a matrix may provide useful information (Lancet 2003; 362(9382): 433-449, Cancer Res 2005; 65(17): 7674-7681, Mol Cell Proteomics 2006; 5: 1975-1983).
A crude extract of Tabernaemontana catharinensis including voacangine has been reported as a powerful anticancer agent (C. G. Pereira, J. E. Carvalho, M. A. A. Meireles, Anticancer activity of Tabernaemontana catharinensis extract obtained by supercritical fluid extraction, Rev. Bras. Pl. Med., Botucatu, V. 8, N. 4 (2006), 144-149). Additionally, voacangine is known as dose-dependently inhibiting capsaicin-induced contraction (M. W. Lo, Matsumoto, M. Iwai, K. Tashima, M. Kitajima, S. Hone, H. Takayama, Inhibitory effect of iboga-type indole alkaloids on capsaicin-induced contraction in isolated mouse rectum, J. Nat. Med. 65 (2011) 157-165).
The inventors found for the first time that voacangine was a new natural small-molecular compound inhibiting in vitro and in vivo angiogenesis by a nontoxic dose. The inventors screened 300 crude extracts which are taken from a natural plant that is effective in proliferating Human Umbilical Vascular Endothelial Cell (HUVEC) by using a cell-based screening technique. As a result of the screening, the inventors found that voacangine as a new natural small-molecular compound is characterized in anti-angiogenesis.
Additionally, since voacangine dose-dependently inhibited expression levels of HIF-1α and VEGF that is a target gene of HIF-1α and thereby inhibited VEGF-induced angiogenic responses without cell toxicity in low concentration through inhibition of expression of angiogenesis inhibition factors, the inventors verified that voacangine could peculiarly disturb an angiogenesis signaling process.
However, in the case that voacangine showing such general functions is injected into a biotissue, there has not been proposed any technology for detection of its presence or absence and for measurement of its distribution state in a cell.